Human phagocytes rely on the NADPH oxidase to transfer electrons from NADPH to molecular oxygen, thereby generating an array of reactive oxygen species that contribute to host defense. Flavocytochrome b558, the essential membrane component of this NADPH oxidase, is a heterodimeric protein composed of p22p'hox and gp91 phox and previously thought to be a protein unique to phagocytes, although recent studies have identified a family of NADPH oxidase proteins (NOX protein family) that shares homology with gp91 phox and includes members that are expressed in non-phagocytes. Within the, family are members that possess both an NADPH oxidase and a peroxidase-like domain (the so-called dual oxidases, or Duox). Many aspects of the structure, biosynthesis, subcellular distribution or function of the NOX and Duox proteins are unknown. We propose studies to examine features of the biosynthesis, structural organization, and subcellular distribution of three members of the NOX protein family and will pursue three specific aims: 1. To define functionally critical features of NOX2-p22phox association: focusing our attention on heterodimer formation, heme acquisition, and quality control of synthesis in the endoplasmic reticulum. 2. To characterize the biosynthesis of NOX3: focusing our attention on determinants of interactions with p22phox and heme acquisition, and the differential species-specific requirements for cytosolic factors. 3. To characterize the biosynthesis of Duox2 and elucidate the biochemical activity of its individual domains: focusing our attention on spectral properties and biochemical activity of two distinct structural domains in Duox2 and assessing quality control during its biosynthesis in heterologous systems. We believe that these studies will provide important insights into the structural organization of specific NOX proteins that may serve as a basis for understanding the respective tissue distribution and targeted function of members of this newly recognized protein family. In addition, the insights obtained from our proposed studies will have direct clinical implications within the context of gene therapy for chronic granulomatous disease due to an absence of gp91 phox/p22phox, and perhaps for disorders of polytopic membrane proteins in general.